Single-stage enzyme-linked immunosorbent assays (ELISA) are commonly used for the qualitative or quantitative determination of an analyte. In this process an analyte is affixed to a surface and an antibody, specific for the analyte, is added. The antibody is covalently linked to an enzyme or can itself be detected by a secondary antibody or streptavidin or avidin, which is linked to an enzyme through bioconjugation. An enzymatic substrate is then added to produce a signal, such as color change, fluorescence or luminescence, which indicates the quantity of analyte in the sample. Since a single enzyme reacts with more than one enzymatic substrate, the signal is amplified and thereby increases the sensitivity of the analysis.
Successive enzymatic reactions can also be employed to increase the amplification effect. A first enzyme is used to generate multiple copies of a second enzyme, which in turn produce multiple copies of a detectable species such as an optical agent, a fluorescent dye, or a luminescent agent. In this manner signal amplification is increased through the use of coupled enzymatic reactions.